Use of the long pentraxin ptx3 for the treatment of diseases caused by an altered activation of the growth factor fgf-2

ABSTRACT

The use of the long pentraxin PTX3 (PTX3) or one of its functional derivatives is described for the preparation of medicament which inhibits the biological activity of the growth factor FGF-2, useful for the prevention and treatment of diseases brought about by an altered activation of said growth factor FGF-2.

[0001] The present invention relates to the use of the long pentraxinPTX3 (PTX3) or one of its functional derivatives for the preparation ofa medicament which inhibits the biological activity of the growth factorFGF-2, said medicament being useful for the prevention and treatment ofdiseases brought about by an altered activation of said growth factorFGF-2.

[0002] Among the diseases brought about by an altered activation of thegrowth factor FGF-2 are included diseases provoked by an alteredangiogenesis and by an uncontrolled proliferation of fibroblasts orsmooth muscle cells.

[0003] The first compound endowed with antiangiogenic activity wasdiscovered in the cartilage by Henry Brem and Judath Folkman in 1975 (J.Exp. Med. Feb. 1, 1975;141(2):427-39). The authors thought that thisdiscovery could be used to control pathological process, such as tumorgrowth, metastasis, chronic and acute inflammation of the joint anddiabetic retinopathy, using selective inhibitors of the pathologicalneoangiogenesis.

[0004] Angiogenesis in the adult is normally quiescent, but itrepresents a normal function, for example in the healing of wounds, orin the reconstruction of the endometrium during the female reproductivecycle.

[0005] The angiogenic response is physiologically stimulated when thevascular functions are reduced and tissue perfusion is inadequate.

[0006] More generally, it can be claimed that, in physiologicalconditions, angiogenesis constitutes a positive feedback in response toinadequate perfusion, or to a reduced supply of oxygen and nutrients,such as occurs, for instance, in the case of occlusion of an artery, insituations of tissue mass growth (for example, the neovascularisationthat accompanies the formation of muscle tissue); and in the case of anincreased work load in association with an increased oxygen and nutrientrequirement.

[0007] It is well known that the growth of a primary tumor is favouredby good vascularisation of the tumor tissue. An adequate supply ofoxygen and nutrients promotes rapid growth of the tumor itself.

[0008] It has been demonstrated that the extent of angiogenesis can bean extremely negative factor in the prognosis of neoplasms (vanHinsbergh V W, Collen A, Koolwijk P; Ann. Oncol., 10 Suppl., 4:60-3,1999; Buolamwini J K; Curr., Opin., Chem., Biol., 3(4):500-9, August1999).

[0009] It is also known, in the tumor field, that a fundamental stage inthe biology of the tumor cell is the acquisition of metastasisingcapability.

[0010] The tumor cells that metastasise are able to lose adherence tothe surrounding structures, invade blood and lymphatic vessels andcolonise other tissues at a distance where they can continue toreproduce themselves.

[0011] Metastasising is also a critical event in the clinical history ofthe disease, being the main cause of death due to cancer. It is closedassociated with and facilitated-by the presence of vascular tissue inthe tumor site or adjacent areas.

[0012] The migration of tumor cells across the surrounding structuresenables the cells to reach the intra-tumor blood vessels, whetherpre-existing or formed by neo-angiogenesis, and thus reach thebloodstream (Ray J M., Stetler-Stevenson W G; Eur. Respir. J.,7(11):2062-72, 1994; Stetler-Stevenson W G, Liotta L A, Kleiner D E Jr;FASEB J., 7(15):1434-41, December 1993).

[0013] The presence of communication between lymphatic and blood vesselsin the vascular region of the tumor enables the neoplastic cells to movein both vascular systems.

[0014] Recent studies have shown a direct relationship betweenangiogenesis and arthritic disease (Koch A E; Arthritis and Rheumatism41:951-962, 1998). In particular, it has been demonstrated thatneo-vascularisation of the articular cartilages plays a crucial role inpannus formation and in progression of arthritis. A normal cartilagedoes not possess blood vessels, while the synovial fluid of arthriticpatients contains an angiogenesis-stimulating factor produced byendothelial cells (EASF).

[0015] The presence of this factor is associated with vascularisationand degradation of the cartilage.

[0016] Other diseases are also related to abnormal angiogenesis.

[0017] It has been found that, in diabetic retinopathy [HistolHistopathol. October 1999; 14(4): 1287-94], psoriasis [Br J Dermatol.December 1999; 141(6):1054-60], chronic inflammation andarteriosclerosis [Planta Med December 1998; 64(8):686-95],neovascularisation of the affected tissues is a facilitating factor.

[0018] The growth factor FGF-2 is a cationic polypeptides of 18 kDacapable both of inducing neoangiogenesis in vivo and cellsproliferation, chemotaxis and production of proteases in endothelialcells in culture (Basilico and Moscatelli, 1992, Adv. Cancer Res.59:115-65).

[0019] The role of FGF-2 in the tumoral angiogenesis is already known(Pathol. Biol. (Paris) April 1999; 47(4):364-7).

[0020] In J. Pathol. September 1999; 189(1):72-8 is reported that anincreased expression of FGF-2, in patients suffering from mesotelioma,is related to an higher aggressiveness and higher mortality of patientsaffected by such tumoral disease.

[0021] In Oncogene Nov. 18, 1999;18(48):6719-24 is reported that FGF-2confers metastasising propriety to rat bladder carcinoma cells.

[0022] Int. J. Cancer May 5, 1999;81(3):451-8 reports that FGF-2 has astrong mitogenic activity, and is involved in the development of humantumors and in the increases of their malignity.

[0023] For the cure of tumoral diseases the antiangiogenic therapyrespect to the standard traditional chemotherapy presents the followingadvantages (Cancer Research 1998, 58, 1408-16):

[0024] 1. Higher specificity: it has as target the tumorneovascularisation process;

[0025] 2. Higher biodisponibility: it has as target the endothelialcells, easy reachable without the problems linked to the traditionalchemotherapeutic approach which act directly on the tumoral cells;

[0026] 3. Minor chemoresistence: this may be the most importantadvantage of this therapy; in fact, being the endothelial cells, respectto the tumoral cells, genetically stable, phenomenon of drug resistanceare improbable;

[0027] 4. Minor metastasising: the blockage of the neovascularisationlimits the propagation of the tumor cells in the other parts of the bodythrough the bloodstream;

[0028] 5. The apoptosis is favoured: the blockage of the vascular net inthe tumor decrease the oxygen and nutrient availability for the tumoralcells, in this way the apoptosis is increased;

[0029] 6. Reduction of the systemic toxicity: toxic effect, such asmielosuppression, gastrointestinal effects and transient alopecia,present with the traditional chemotherapy, are not observed with theantiangiogenic therapy.

[0030] To determinate these effects FGF-2 reacts with two differentreceptor classes present on target cells surfaces: the receptors havinghigh affinity endowed with tirosin kinases activity (FGFRs) andreceptors endowed with low affinity represented by proteoglycan eparansulfate (HSOGs) (Johnson and Williams, 1993, Adv. Cancer Res. 60:1-41;Moscatelli, 1987, J. Cell. Physiol. 131:123-30).

[0031] FGF-2 is a potent mitogen for medial smooth muscle cells and isnecessary for their proliferation after balloon catheter injury (J.Biol. Chem. Apr. 14, 2000; 275 (15): 11270-7).

[0032] Therefore, diseases caused by an altered activation of the growthfactor FGF-2 also include the hyperplasia of the muscular wall of thearteries that occurs during restenosis after angioplastic or “coronarystenting” [J. Vasc. Surg. February 1997; 25(2):320-5].

[0033] The control of FGF-2 dependent neovascularisation represents oneof the fundamental elements for the control and cure of diseases linkedto an altered angiogenesis, as well as the control of FGF-2-dependentuncontrolled proliferation of fibroblasts or SMCs is crucial for thetreatment of cicatrization linked to excessive fibroblastic response andrestenosis after angioplastic.

[0034] The availability of new therapeutical compound which specificallyinhibit the biological activity of FGF-2 is an objective of primaryimportance for the prevention and treatment of diseases brought about byan altered activation of this growth factor. Such diseases includearthritic disease, tumor, tumor metastasis, diabetic retinopathy,psoriasis, chronic inflammation, arteriosclerosis, cicatrization linkedto excessive fibroblastic response and restenosis after angioplastic.

[0035] PTX3 is expressed in various cell types (Bottazzi et Al., J.,Biol. Chem. 1997, 272: 32817-32823) particularly in mononuclearphagocytes and endothelial cells, after exposure to the inflammatorycytokines Interleukin 1β (IL-1β) and Tumor Necrosis Factor alpha(TNF-α).

[0036] To date, the biological function of PTX3 has not yet been fullyunderstood.

[0037] PTX3 consists of two structural domains, a N-terminal domainunrelated to any known molecule, and a C-terminal domain similar to theshort pentraxins such as C-reactive protein (CRP) (Breviario F., et al.,J. Biol. Chem. 267:22190, 1992).

[0038] A substantial degree of similarity has been found between humanPTX3 (hPTX3) and animal PTX3s. In particular, mouse PTX3. (mPTX3) isvery similar to hPTX3 in terms of DNA sequence and gene organisation andlocation. The degree of identity between human and murine PTX3 gene is82%, and reaches 90% if conservative substitutions are considered(Introna M., et al.: Blood 87 (1996) 1862-1872). The murine PTX3 gene islocated on chromosome 3 of the mouse in a region similar to the human 3qregion (q24-28), in agreement with the documented location of hPTX3 inthe 3q 25 region (Introna M., et al.: Blood 87 (1996) 1862-1872).

[0039] The high degree of similarity between hPTX3 and mPTX3 sequencesis a sign of the high degree of conservation of pentraxins duringevolution (Pepys M. B., Baltz M. L.: Adv. Immunol. 34:141, 1983).

[0040] For a review about pentraxins see H. Gewurz et al, CurrentOpinion in Immunology, 1995, 7:54-64.

[0041] The international application WO99/32516, filed in the name ofthe applicant, describes the use of the long pentraxin PTX3 for thetreatment of infective diseases, inflammatory or tumoral. The anti-tumoractivity shown by PTX3, described in WO99/32516, is mediated by theleucocitary recruitment, i.e. on immunologic bases. In WO99/32516 it isnever described or suggested the use of PTX3 as useful agent for thetreatment of diseases associated with an altered activation of thegrowth factor FGF-2.

[0042] U.S. Pat. No. 5,767,252 describes a neuronal growth factorbelonging to the pentraxin family (see also the references there cited).This patent refers to the neurobiology sector.

[0043] It has now been found that the long pentraxin PTX3 is capable tobind the FGF-2 with high affinity and specificity. The binding of PTX3with FGF-2 (K_(d)=8-16 nM) prevents the binding of the latter to itshigh affinity tyrosine-kinase receptors, and the binding at the site oflow affinity receptors, represented by the eparan-sulphateproteoglycans, present on the cell surface. The inhibition of thebinding causes an inhibition of the FGF-2 biological activity.

[0044] The interaction between FGF-2/PTX3 depends on a correctconformational structure of the growth factor, not only on its basicnature, since PTX3 does not bind the cytocrome C (a protein that sharewith the FGF-2 molecular weight (18 kDa) and isoelectric point (pH9.6)).

[0045] Moreover, the C-reactive protein, homologous to PTX3, does notbind FGF-2.

[0046] Is therefore object of the present invention the use of the longpentraxin PTX3 or a derivatives thereof, or its domain, for preparing amedicament for the prevention and cure of diseases which arecounteracted by the inhibition of the biological activity of the growthfactor FGF-2.

[0047] A further object of the present invention is the use of the longpentraxin PTX3 or a derivatives of the PTX3 or its domain, for preparinga medicament for the prevention and cure of diseases brought about by analtered activation of the growth factor FGF-2. A further object of thepresent invention is the use of the long pentraxin PTX3 or a derivativethereof, or its domain, for preparing a medicament for the prevention orcure of diseases brought about by an altered angiogenesis, in which thealtered angiogenesis is provoked by an altered activation of the growthfactor FGF-2, example of said diseases are: arthritic disease, tumormetastasis, diabetic retinopathy, psoriasis, chronic inflammation,arteriosclerosis or tumor, in which the tumor is, for example, sarcoma,carcinoma, carcinoid, bone tumor or neuroendocrine tumor.

[0048] A further object of the present invention is the use of the longpentraxin PTX3 or a derivatives of the PTX3 or its domain, for preparinga medicament for the treatment of diseases associated with uncontrolledFGF-2-dependent proliferation of fibroblasts or smooth muscular cells,such as the cicatrization linked to excessive fibroblastic response, andthe restenosis after angioplastic.

[0049] The compound according to the invention is suitable to be usedfor the inhibition of FGF-2 activity in target cells not only when it isadministered as recombinant protein, but also when it is administeredendogenously in consequence of the gene transfer of its cDNA.

[0050] Is therefore a further object of the present invention the use ofcDNA full length of human PTX3 or its derivative or its domain, forpreparing plasmidic or viral expression vectors comprising said cDNA,for the gene therapy of diseases caused by an altered activation of thegrowth factor FGF-2, in which the diseases are, for example, tumor,tumor metastasis, cicatrization linked to excessive fibroblasticresponse or restenosis after angioplastic.

[0051] For “long pentraxin PTX3” is intended any long pentraxin PTX3,independently from its origin natural (human or animal) or synthetic.

[0052] For derivatives is intended any functional analogous of the longpentraxin PTX3 as above defined that bear at least a mutation,maintaining its functional capacity to selectively inhibit the FGF-2.

[0053] The preferred long pentraxin PTX3 is the human PTX3, whichsequence is described in WO99/32516.

[0054] The long pentraxin PTX3 described herein can also be used incombination with one or more anticancer drugs for the treatment of tumordiseases in which the increased expression of FGF-2 provokes an higheraggressiveness of the tumor disease, or an higher metastasisingcapability.

[0055] In fact, it is well known that, to avoid the unwanted sideeffects maintaining the same therapeutic efficacy, most oncologicalpatients are treated not with a single anticancer drug, but with acombination of several anticancer agents or with a combination ofanticancer agents in association with antiangiogenic compounds.

[0056] The mechanism of action of the more usual anticancer drugs iscompletely different from the mechanism of action of the compoundaccording to the present invention, in fact, the usual anticancer drugsare cytotoxic vs tumoral cells.

[0057] The compound according to the present invention having adifferent mechanism of action, exert a curative effect (adjuvant effect)additional to the effect exerted by the anti-tumor drug to which it isassociated.

[0058] It is therefore a further object of the invention describedherein the combination of the long pentraxin PTX3 with one or more knownanticancer drugs.

[0059] A further object of the invention described herein is apharmaceutical composition containing the long pentraxin PTX3 incombination with one or more known anticancer drugs, such as alkylatingagents, topoisomerase inhibitors, antitubulin agents, intercalatingcompounds, anti-metabolites, natural products such as vinca alkaloids,epipodophyllotoxins, antibiotics, enzymes, taxans, andcyto-differentiating compounds, and one or more excipients or vehiclespharmacologically acceptable.

[0060] A further object of the invention described herein is the use ofthe long pentraxin PTX3, in combination with one or more knownanti-tumor compounds, for preparing a medicament for the treatment oftumor, in which the increased expression of FGF-2 provokes an higheraggressiveness of the tumor.

[0061] A further object of the invention described herein is the use ofthe long pentraxin PTX3, in combination with one or more knownanti-tumor compounds, for preparing a medicament for the prevention ofthe onset of tumor metastasis, in which the increased expression ofFGF-2 provokes an higher metastasising capability.

[0062] A further object of the invention described herein is the use ofthe long pentraxin PTX3 in combination with, the anticancer compound,for preparing a medicament for the treatment of tumor, characterised inthat the long pentraxin PTX3 is present as adjuvant of the anticancercompound.

[0063] The following examples illustrate the invention.

EXAMPLE 1

[0064] Capacity of PTX3 to Bind FGF in Solution.

[0065] This experiment has been performed in order to evaluate PTX3binding to FGF-2. PTX3 has been produced as described by Bottazzi etal., 1997, J. Biol. Chem. 272:32817-32823. Human recombinant FGF-2 hasbeen produced as described by Isacchi A. et al., Proc. Natl. Acad. Sci.U.S.A. (1991), 88:2628-32. When necessary, FGF-2 has been labelled with¹²⁵I following the method described by Isacchi et al. (see below).

[0066] Experimental Procedure:

[0067] 100 μl of PBS containing 1 μg of FGF-2, (or alternatively 1 μg ofPTX3) were chromatographed on a size exclusion fast protein liquidchromatography Superose 12 column (Pharmacia): this column is able toseparate proteins depending on their molecular weight. The column waseluted with PBS at a flow rate equal to 1 ml/minute and 1 ml fractionswere collected and analyzed by dot, blot with specific antibodies forthe two proteins. In order to evaluate PTX3 binding to FGF-2 the twoproteins (5 g and 1 μg, respectively), were mixed in 100 μl PBS andincubated at 4° C. for 10 min. before loading onto the column. Fractionswere collected and analysed by dot-blot with anti FGF-2 specificantibodies. The results reported in FIG. 1 show that FGF-2 (18.000 D) iseluted from the column with a retention volume of approximately 22 ml.In the same conditions PTX3 (450.000 D) is eluted with the void volumeof the column (7 ml.) When FGF-2 pre-incubated with PTX3 for. 10 min. at4° C. was loaded onto the column, a change in its chromatographicbehaviour was observed: in this experimental conditions FGF-2 was elutedfrom the column with a retention volume equal to PTX3 alone.

EXAMPLE 2

[0068] Capacity of Biotinylated PTX3 to Bind Plastic Immobilised FGF-2

[0069]100 μl of NaHCO₃ pH 9.6 containing 500 ng of FGF-2 or of thecomplement component C1q were incubated 18 h at 4° C. in 96 wellsplastic plates. At the end of incubation wells were washed 3 times withPBS and subsequently incubated 2 h at room temperature with 200 μl ofPBS containing 1 mg/ml of Bovine Serum Albumin (BSA). At the end ofincubation wells were washed again 3 times with PBS and incubated with30 ng/ml of ¹²⁵I-FGF-2 in presence of increasing amounts of biotinylatedPTX3 in 100 μl of PBS (2 h at room temperature). Alternatively wellswere adsorbed with increasing doses of FGF-2 or C1q and incubated with100 ng/ml of biotinylated PTX3 in 100 μl of PBS. After this incubationwells were washed 3 times with PBS and incubated 1 h at room temperaturewith 100 μl of streptavidin HRP conjugate ({fraction (1/2000)}).Reaction was developed by adding the chromogen microwell peroxidasesubstrate system ABTS (Kirkegaard & Perry Laboratories). Plates wereread in an automatic ELISA reader at 405 nm. Results reported in FIG. 2demonstrate that FGF-2 immobilised on plastic wells is able to bindbiotinylated PTX3 in a way similar to the physiologic PTX3 ligand C1q(Bottazzi B. et al., 1997, J. Biol. Chem. 272:32817-32823).

EXAMPLE 3

[0070] Characterisation of PTX3-FGF-2 Interactions

[0071] 96 wells plates were coated at 4° C. with 100 μl of NaHCO₃ pH 9.6containing PTX3 or C reactive protein (200 nM) or alternatively 2 μg/mlof the following proteins used as negative control: bovine serumalbumin, fibronectin, gelatine or laminin. After incubation, wells werewashed 3 times with PBS and blocked 2 h at room temperature with 200 μlof 1 mg/ml of BSA in PBS. At the end of incubation wells were washed 3times with PBS and subsequently incubated with 30 ng of ¹²⁵I-FGF-2 for 2h at room temperature. Wells were subsequently washed 3 times with PBSand bound ¹²⁵I-FGF-2 was recovered by washing each well twice with 200μl of 2% SDS in water. Levels of ¹²⁵I-FGF-2 were measured in a gammacounter. Results reported in FIG. 3 shows that FGF-2 binds plasticimmobilised PTX3 and that this binding is specific since FGF-2 reactspoorly with CRP and does not react with other immobilised proteins.

EXAMPLE4

[0072]¹²⁵I-FGF-2 Binding to Plastic Immobilised PTX3 in the Presence ofan Excess of Cold FGF-2

[0073] In a second set of experiments the binding of ¹²⁵I-FGF-2 toplastic immobilised PTX3 has been tested in the presence of an excess ofboth native or heat denatured cold FGF-2 (100 nM), in the presence ofsimilar Cytocrome C concentrations or in the presence of an excess ofsoluble PTX3 (300 nM). The experiment was performed as described above:The results reported in FIG. 4 demonstrate that cold FGF-2 and solublePTX3 inhibited the interaction between ¹²⁵I-FGF-2 and plasticimmobilised PTX3. The observation that heat-inactivated FGF-2 andCytocrome C (having the same molecular weight and isoelectric point asFGF-2) are not able to bind PTX3 suggests that a correct conformation ofthe growth factor rather than its basic nature is involved in itscapacity to bind PTX3.

EXAMPLE 5

[0074] Dissociation Constant (Kd) of FGF-2/PTX3 Interaction

[0075] With this experiment the dissociation constant (Kd) FGF-2/PTX3interaction has been determined. For this purpose increasing doses of¹²⁵I-FGF-2 were incubated with plastic immobilised PTX3 and bindingresults were analysed by Scatchard plot. Results reported in FIG. 5demonstrate that FGF-2 binds PTX3 in a saturable way and with elevatedaffinity (Kd=8-16 nM). This affinity is similar to the affinitypreviously calculated for the interaction of PTX3 with its physiologicalligand C1q.

EXAMPLE 6

[0076] Effect of PTX3 on FGF-2 Binding to Endothelial Cells

[0077] Transformed foetal bovine aortic endothelial cells GM7373 wereseeded at 80,000 cells/cm² in 24-well dishes in Eagle's minimalessential medium (Eagle's MEM) containing 10% FCS. After 24 h at 37° C.adherent cells were washed twice with Eagle's MEM without FCS andsubsequently incubated for 2 h at 4° C. with ¹²⁵I-FGF-2 (10 ng/ml) inEagle's MEM containing 0.15% gelatine and 20 mM HEPES pH 7.5 in theabsence or in the presence of increasing concentrations of PTX3. At theend of incubation the amount of ¹²⁵I-FGF-2 bound to low (HSPGs) and highaffinity receptors (FGFR) was evaluated as described by Moscatelli, 1987J. Cell. Physiol. 131:123-30. Briefly cells were rinsed twice with 2 MNaCl in 20 mM HEPES buffer (pH 7.5) to remove ¹²⁵I-FGF-2 bound tolow-affinity binding sites and twice with 2 M NaCl in 20 mM sodiumacetate (pH 4.0) to remove ¹²⁵I-FGF-2 bound to high-affinity bindingsites. Non-specific binding was measured in the presence of a 100-foldmolar excess of unlabeled FGF-2 and subtracted from all the values.Results reported in FIG. 6 shown that PTX3 is able to inhibit in adose-dependent manner the binding of FGF-2 to its high- and low-affinityreceptors on endothelial cells.

EXAMPLE 7

[0078] Effect of PTX3 on Mitogenic Activity Exerted by FGF-2 onEndothelial Cells

[0079] GM 7373 cells were seeded at 75,000 cells/cm² in 48-well platesin Eagle MEM containing 10% FCS. After 24 h at 37° C. adherent cellswere washed twice with Eagle's MEM without FCS and subsequentlyincubated for 24 h at 37° C. in Eagle's MEM containing 0.4% FCS in theabsence or in the presence of FGF-2 (10 ng/ml) and increasingconcentrations of PTX3. At the end of incubation cells were trypsinizedand counted. In a different set of experiments GM 7373 cells weretreated as described above in the presence of different mitogenicstimuli: 10% FCS; 5 μg/ml dyacyl-glycerol (DAG); 10 ng/ml phorbol ester(TPA) 30 ng/ml epidermal growth factor (EGF) or 30 ng/ml vascularendothelial growth factor (VEGF). Results reported in FIG. 7 shown thatPTX3 inhibits the mitogenic activity exerted on endothelial cells byFGF-2 in a dose-dependent manner with an ID₅₀ equal to 30 nM. This valueis similar to the Kd calculated for the interaction FGF-2-PTX3. Theseresults suggest that the inhibition of FGF-2 biological activity by PTX3is due to its sequestration in extracellular sites. The inhibitoryactivity of PTX3 on FGF-2 is specific since it is not detectable whenother mitogens are used to induce, cellular proliferation.

EXAMPLE 8

[0080] Effect of PTX3 on the Proliferation of Transformed Foetal BovineAortic Endothelial Cells

[0081] In this experiment we have investigated the effect of PTX3 on theproliferation of the murine aortic endothelial cell line MAE3F2T stablytransfected with an expression vector coding for FGF-2 (Gualandris etal. 1996, Cell Growth Diff. 7:147-60). These cells can proliferate inresponse to a autocrine loop of stimulation induced by endogenous FGF-2(Gualandris et al. 1996, Cell Growth Diff. 7:14760). MAE3F2T cells wereseeded at 10,000/cm² in 48-well plates in Dulbecco medium (DMEM) addedwith 10% FCS. After 24 h at 37° C., adherent cells were washed twicewith DMEM without FCS and incubated for 72 at 37° C. in DMEM containing0.4% FCS in the presence or not of PTX3 (70 nM), anti FGF-2 specificantibodies or suramin (50 μg/ml). Suramin is known for its capacity tosequestrate extracellular FGF-2 and inhibits its biological activities(Rusnati et al., 1996, Mol. Biol. Cell. 7:369-381). At the end ofincubation cells were trypsinized and counted in a Burker chamber.Results reported in FIG. 8 shown that PTX3 is able to block theautocrine loop of stimulation induced by FGF-2 in MAE3F2T cells,inhibiting their FGF-2 dependent proliferation, in a manner similar tothat exerted by anti FGF-2 antibodies and suramin.

EXAMPLE 9

[0082] Effect of PTX3 onto the Neovascularization Induced In Vivo byFGF-2

[0083] The antiangiogenic potential of PTX3 was evaluated in vivo in thechick embryo chorioallantoic membrane (CAM) assay. Briefly, a window wasopened in the egg shell of 3 day-old fertilized chicken eggs. At day 8,gelatin sponges were implanted on the CAMs and adsorbed with 10 ul ofPBS alone or containing FGF-2 (at 500 ng/sponge) in the absence or inthe presence of PTX3 (5 ug/sponge) (5-6 embryos per group). After 4days, CAMs were observed in ovo under a Zeiss SR stereomicroscope andthe angiogenic response was scored by two investigators withoutknowledge of the samples tested and graded on an arbitrary scale of0-4+, with 0 representing no angiogenic response and 4+ representing thestrongest activity. Results reported in FIG. 9 shown that PTX3 is ableto block the neovascularization induced in vivo by FGF-2.

EXAMPLE 10

[0084] Gene Therapy with PTX3

[0085] Murine endothelial cells overexpressing FGF-2, named FGF2MAE3F2T(Gualandris et al. 1996, Cell Growth Diff. 7:147-60) were transfectedwith human PTX3 full length cDNA subcloned in the commercial expressionvector pLXSH (Clontech).

[0086] In vitro studies on the transfected cell lines obtained were,performed to study the effect of PTX3 over-expression on FGF-2-dependentproliferation of FGF2MAE3F2T cells and their invasive behaviour onthree-dimensional fibrin gels.

[0087] In details, several FGF2MAE3F2T, clones expressing differentlevels of PTX3 and parental FGF2MAE3F2T cells (that do not produce PTX3)were seeded at 10,000/cm² in 48-well plates in Dulbecco medium (DMEM)added with 10% FCS. After 24 h at 37° C., adherent cells were washedtwice with DMEM without FCS and incubated for different period of timeat 37° C. in DMEM containing 0.4% FCS. At the end of incubations cellswere trypsinized and counted in a Burker chamber. Results reported inFIG. 10, shown that PTX3 over-expression inhibits FGF2MAE3F2T cellsproliferation and that the extent of this inhibition correlates with theamount of PTX3 produced by the different clones tested.

[0088] To evaluate the invasive behaviour of FGF2MAE3F2T clonesover-expressing PTX3 on three-dimensional fibrin gels, cell aggregateswere prepared on agarose-coated plates exactly as described (Gualandriset al. 1996, Cell Growth Diff. 7:147-60). These aggregates were seededonto fibrin-coated 48 well-plates. Immediately after seeding, 250 l ofcalcium-free medium containing fibrinogen (2.5 mg/ml) and thrombin (250mU/ml) were added to each well and allowed to gel for 5 min at 37° C.Then, 500 l of culture medium were added on the top of the gel. In allthe, experiments, the fibrinolytic inhibitor trasylol was added to thegel and to the culture medium at 200 KIU/ml to prevent the dissolutionof the substrate. Formation of radially growing cell sprouts wasevaluated after 2 days by computerized image analysis.

[0089] Results reported in FIG. 11 shown that the invasive capacity ofPTX3 over-expressing FGF2MAE3F2T clones is significantly lower that thatof parental FGF2MAE3F2T cells.

[0090] The results obtained demonstrate that PTX3 inhibits FGF-2activity in endothelial cells when it is endogenously produced aftergene transfer of its cDNA.

[0091] Thus, the compound according to the invention can be used in genetherapy protocols in accordance, to known methods (In Vivo.January-February 1998; 12(1):59-67; In Vivo. January-February 1998;12(1):35′-41; In Vivo. November-December 1994; 8(5):771-80) for thetreatment of diseases caused by an altered activation of the growthfactor FGF-2.

1. Use of the long pentraxin PTX3 or a derivative thereof or its domain,for preparing a medicament for the prevention and cure of diseasesbrought about by an altered activation of the growth factor FGF-2, inwhich said disease is selected from the group consisting of:uncontrolled proliferation of fibroblasts or smooth muscle cells, or analtered angiogenesis.
 2. Use according to claim 1, in which the diseasecaused by an uncontrolled proliferation of fibroblasts or smooth musclecells, or by an altered angiogenesis is selected from the groupconsisting of: cicatrization linked to excessive fibroblastic response,restenosis after angioplastic, arthritic disease, diabetic retinopathy,psoriasis and atherosclerosis.
 3. Use according to claims 1 or 2, inwhich the long pentraxin PTX3 is the PTX3 present in nature.
 4. Useaccording to claim 3, in which the long pentraxin PTX3 is the humanPTX3.
 5. Use according to claims 1-2, in which the long pentraxin PTX3is the PTX3 from synthetic origin.
 6. Use of the cDNA that code for thelong pentraxin PTX3 or its derivative or its domain, for preparingexpression vectors comprising said cDNA, for the gene therapy ofdiseases caused by an altered activation of the growth factor FGF-2, inwhich the disease is selected from the group consisting of anuncontrolled proliferation of fibroblasts or smooth muscle cells, or analtered angiogenesis.
 7. Use according to claim 6, in which said cDNAcontaining the gene coding for PTX3 or its derivative is carried by aplasmidic or viral vector.
 8. Use according to claim 6, in which thedisease caused by an uncontrolled proliferation of fibroblasts or smoothmuscle cells, or by an altered angiogenesis is selected from the groupconsisting of cicatrization linked to excessive fibroblastic response,restenosis after angioplastic, arthritic disease, diabetic retinopathy,psoriasis, and atherosclerosis.
 9. Use of the long pentraxin PTX3 forpreparing a medicament useful for blocking or decreasing theaggressiveness of a tumor, in which said aggressiveness is caused by anincreased expression of FGF-2.
 10. Use of the long pentraxin PTX3 forpreparing a medicament for the prevention of the onset of tumormetastasis, in which the increased expression of FGF-2 provokes anhigher metastasising capability.
 11. Use according to claims 9 or 10, inwhich the tumor is selected from the group consisting of sarcoma,carcinoma, carcinoid, bone tumor and neuroendocrine tumor.
 12. Useaccording to claims 9-11, in which the long pentraxin PTX3 is incombination with one or more known anticancer drugs, characterised inthat said long pentraxin PTX3 is present as adjuvant of the anticancerdrug.